QC Report


general
Report generated at2025-02-25 03:26:49
TitleAdrenalGland
DescriptionATAC-seq data from alternative Human ENCODE adrenal gland data processed in 2025
Pipeline versionv2.2.0
Pipeline typeatac
Genomehg38
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads68530869264080026
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads67528141063376096
Mapped Reads (QC-failed)00
% Mapped Reads98.598.9
Paired Reads68530869264080026
Paired Reads (QC-failed)00
Read134265434632040013
Read1 (QC-failed)00
Read234265434632040013
Read2 (QC-failed)00
Properly Paired Reads66479184262244114
Properly Paired Reads (QC-failed)00
% Properly Paired Reads97.097.1
With itself67146573063057354
With itself (QC-failed)00
Singletons3815680318742
Singletons (QC-failed)00
% Singleton0.60.5
Diff. Chroms20273213939
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads29409835023557643
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads10725456110263652
Paired Optical Duplicate Reads10732565312
% Duplicate Reads36.468943.568200000000004

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads55284942032888947
Rm = Number of Mitochondrial Reads13370366633619613
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.194746289436968620.5054930222515718

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads37041039224993228
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads37041039224993228
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads37041039224993228
Paired Reads (QC-failed)00
Read118520519612496614
Read1 (QC-failed)00
Read218520519612496614
Read2 (QC-failed)00
Properly Paired Reads37041039224993228
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself37041039224993228
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.5846580713564120.6681063190700142
Fraction of reads in NFR (QC pass)TrueTrue
Fraction of reads in NFR (QC reason)OKOK
NFR / mono-nuc reads2.42071234401634033.2122539176994622
NFR / mono-nuc reads (QC pass)FalseTrue
NFR / mono-nuc reads (QC reason)out of range [2.5, inf]OK
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakTrueTrue

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.64997297376041220.2775773501526093
Fraction of Reads in blacklist regions0.00051925109055795610.002469909048963183
Fraction of Reads in promoter regions0.31723936082225250.1002065439486248
Fraction of Reads in enhancer regions0.39332519860835870.2870773235053911

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments24628572812897991
Distinct Fragments19161367312504946
Positions with Two Read26182209314948
NRF = Distinct/Total0.7780140.969527
PBC1 = OneRead/Distinct0.8149040.974088
PBC2 = OneRead/TwoRead5.96385338.675975

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt13281844366
N1277262190293
N27693430350
Np276831190049
N optimal276831190049
N conservative13281844366
Optimal Setpooled-pr1_vs_pooled-pr2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio2.08428827417970464.283663165487085
Self Consistency Ratio3.60389424701692376.26995057660626
Reproducibility Testfailfail

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks299736218761

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile297.0154.0502.0334.0
50 percentile (median)543.0215.0776.0590.0
75 percentile932.0353.01159.0978.0
Max size3603.02122.03598.03598.0
Mean677.8524968639069320.4930814907593872.405390188846717.5627621184044

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment26.08425363463648214.011771178374902

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.098185364639049440.22365774548383893
Synthetic AUC0.497985664937452050.49205019167743685
X-intercept0.104025893021358950.21046876124523203
Synthetic X-intercept0.04.6889282558342433e-135
Elbow Point0.87112264399894450.6257666749298297
Synthetic Elbow Point0.50297212364632120.5136372569637959
Synthetic JS Distance0.63743580776433210.3230172010275505

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.60188434454074390.178483027482484460.59547327711043270.227243235647672240.59500681611546150.22824854796667320.5753311869021330.56909824447231920.5686598772160963

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.446730118454656540.59407092984583430.11502207718026660.567628799149588

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.283122430694994650.54974137982608220.074604688918134140.5246375766615389

For macs2 raw peaks:


For overlap/IDR peaks: